The glycosaminoglycans such as heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid are biopolymers extracted industrially from various animal organs.
In particular, heparin, mainly obtained by extraction from the intestinal mucous membrane of pigs or from bovine lung, is a polydispersed copolymer with a molecular weight distribution from approximately 3,000 to approximately 30,000 D consisting of a chain mixture basically consisting of an uronic acid (glucuronic acid or iduronic acid) and of an amino sugar (glucosainine) linked by α-1→4 or β-1→4 bonds. In heparin, the uronic unit can be O-sulfated in position 2 and the glucosamine unit is N-acetylated or N-sulfated, 6-O-sulfated and 3-O-sulfated in approximately 0.5% of the glucosainine units present.
The properties and the natural biosynthesis of heparin in mammals have been described by Lindahl et al., 1986 in Lane and Lindahl (Editors) “Heparin. Chemical and Biological Properties; Clinical Applications”, Edward Arnold, London, Pages 159-190, by Lindahl et al. TIBS, 1986, 11, 221-225 and by Conrad “Heparin Binding Proteins”, Chapter 2: Structure of Heparinoids. Academic Press, 1998. The biosynthesis of heparin occurs starting with its N-acetyl-heparosan precursor formed by a chain mixture consisting of the repetitive glucuronyl-α-1→4-N-acetylglucosamine disaccharide unit. Said precursor undergoes enzymatic modifications which partially hydrolyse the N-acetyl group, substituting it with an SO3-group, epimerize the carboxy in position 5 of a part of the glucuronic units transforming them into iduronic units and introducing O-sulfate groups to get a product which, once extracted industrially, has approximately double the number of sulfate groups as regards carboxy groups per disaccharide unit. These enzymatic modifications lead, i.a. to the formation of the pentasaccharide binding for antithrombin III (ATIII), called active pentasaccharide, which is the structure necessary for the high affinity bond of heparin to ATIII and fundamental for the anticoagulant and antithrombotic activity of the heparin itself. This pentasaccharide, present inside only some of the chains which form heparin, contains a sulfated glucosamine unit in position 3 and a glucuronic acid spaced out between disaccharides containing iduronic acids.
In nature, the formation of the active pentasaccharide is made possible by the epimerization reaction of the carboxy of a part of the glucuronic units into iduronic units carried out by the glucuronyl-C5-epimerase (C5-epimerization) and by suitable sulfation which also leads to the introduction of a sulfate group on the hydroxyl in position 3 of the glucosamine. More particularly, in nature the formation of the active pentasaccharide is made possible by the fact that C5-epimerization occurs in clusters, i.e., on portions of chains, and extensively which leads to a product which contains more iduronic units than glucuronic ones. In fact, commercial heparin contains approximately 70% of iduronic units and 30% of glucuronic units.
Alongside the main anticoagulant and antithbrombotic activities, heparin also exerts antilipaemic, antiproliferative, antiviral, antitumor and antimetastatic activities, but its use as a drug is hindered by the side effects due to the anticoagulant action which can cause bleeding.
It is known that the capsular K5 polysaccharide isolated from Escherichia coli, described by Vann et al., Eur. J. Biochem., 1981, 116, 359-364 (“Vann 1981”), is formed by a mixture of chain consisting of the repetitive disaccharide unit glucuronyl-β-1→4-N-acetyl glucosamine and therefore shows the same repetitive sequence (A)
of N-acetyl-heparosan precursor of heparin. The capsular 1(5 polysaccharide, referred to hereafter as “K5 polysaccharide” or more simply “K5”, was chemically modified by Lormeau et al. as described in U.S. Pat. No. 5,550,116 and by Casu et al., Carbohydrate Res., 1994, 263, 271-284 (“Casu 1994”). K5-O-sulfates having antitumor, antimetastatic, antiviral, in particular anti-HIV activities are described in EP 333243 and WO 98/34958. The K5 was also chemically and enzymatically modified in order to obtain products having in vitro biological activity on coagulation of the same type as that of heparin as extracted from animal organs (extractive heparin).
The attainment of the products having an activity on coagulation of the same type as that of extractive heparin occurs by processes which imitate that occurring in nature and all envisage the key step of C5-epimerization with D-glucuronyl C5 epimerase (Naggi et al., Seminars in Thrombosis and Hemostasis, 2001, 27, 437-443).
The processes described in IT 1230785, WO 92/17507, WO 96/14425 and WO 97/43317 utilize K5 as starting material. The K5 originating from fermentation is subjected to N-deacetylation followed by N-sulfation and on the K5-N-sulfate thus obtained C5-epimerization with C5-epimerase in solution is performed, obtained either by chromatography of a solution of microsomal enzymes from mouse mastocytoma (IT 1230 785) or from bovine liver (WO 92/17507, WO 96/14425 and WO 97/43317).
The D-glucuronyl C5 epimerase from bovine liver was purified by Campbell et al., J. Biol. Chem., 1994, 269, 26953-26958 (“Campbell 1994”) who also provided its composition in amino acids and described its use in solution for the transformation of a K5-N-sulfate into the corresponding 30% epimerized product, demonstrating the formation of iduronic acid by HPLC method followed by total nitrous depolymerization to disaccharide.
The document WO 98/48006 describes the DNA sequence which codes for the D-glucuronyl C5 epimerase and a recombinant D-glucuronyl C5 epimerase, obtained from a recombinant expression vector containing said DNA, afterwards purified by Campbell et al. as shown by Jin-Ping et al., J. Biol. Chem., 2001, 276, 20069-20077 (“Jin-Ping 2001”).
The complete sequence of the C5-epimerase was described by Crawford et al., J. Biol. Chem., 2001, 276, 21538-21543 (“Crawford 2001”).
Beside the key step of C5-epimerization, and immediately after said epimerization, the most recent processes include an oversulfation step of the epiK5-N-sulfate, followed by controlled desulfation of the intermediate oversulfated product thus obtained, giving rise to N-desulfated products as it happens in the case of LMW-heparin (Naggi et al., Carbohydrate Res., 2001, 336, 283-290).
Thus, WO 01/72848 describes a method for the preparation of N-deacetylated N-sulfated derivatives of K5 polysaccharide, at least 40% epimerized to iduronic acid as regards the total of the uronic acids, having a molecular weight from 2,000 to 30,000, containing from 25 to 50% of high affinity chains for ATIII and having an anticoagulant and antithrombotic activity expressed as HCII/antiXa ratio from 1.5 to 4. Said document describes the oversulfation of a 40-60% epimerized K5-N-sulfate and shows that the product obtained, whose 13C-NMR is illustrated, has a sulfate group content per disaccharide unit of 2-3.5. Repeating the aforesaid oversulfation in the conditions described and examining the 13C-NMR it is ascertained that the product obtained is actually a free amine whose content of 6-O-sulfate is 80-95%, that of 3-O-sulfate on the amino sugar is 30%, but whose sulfation degree is 3.2. It is also ascertained that in the conditions of oversulfation described in WO 01/72848 a sulfation degree of more than 3.2 is not obtained.
The Italian patent application MI2001A/00397 (see also WO 02/068477), describes K5-N,O-oversulfates having a sulfation degree of more than 3.2, obtained starting from a K5 polysaccharide free of lipophilic substances or from a fraction thereof with molecular weight of approximately 5,000 by (a) N-deacetylation/N-sulfation, (b) O-oversulfation and, optionally, (c) N-resulfation.
U.S. Pat. No. 7,268,122 and Vicenzi et al., AIDS, 2003, 17, 177-181 disclose the anti-HIV activity of these K5-N,O-oversulfates. According to Vicenzi et al., the tested K5-N,O-oversulfate is more active than K5-O-oversulfate and much more active than heparin.
None of the aforesaid documents describes LMW-K5-N-sulfates, optionally 40-60% epimerized, in which NH2 or acetyl groups are virtually absent.
In order to standardize the terminology and render the text more comprehensible, in the present description conventional terms or expressions will be used, in the singular or plural. In particular:                “K5” or “K5 polysaccharide” designates the capsular polysaccharide from Escherichia coli obtained by fermentation, i.e., a chain mixture consisting of disaccharide units (A) optionally containing a double bond at the non-reducing end as shown above, in any case prepared and purified according to the methods described in literature, in particular according to Vann 1981, according to Manzoni et al., Journal of Bioactive Compatible Polymers, 1996, 11, 301-311 (“Manzoni 1996”) or according to the method described in WO 01/72848 and in WO 02/068447; it is obvious for a person skilled in the art that what is shown hereafter can be applied to any N-acetylheparosan;        “C5-epimerase” designates the D-glucuronyl C-5 epimerase, extractive or recombinant, in any case prepared, isolated and purified, in particular as described in Campbell 1994, in WO 98/48006, in Jin-Ping et al., J. Biol. Chem., 2001, 276, 20069-20077 (“Jin-Ping 2001”) or in Crawford 2001;        “K5-amine” designates at least 95% N-deacetylated K5, but generally in which acetyl groups are undetectable by a current NMR apparatus;        “K5-N-sulfate” designates at least 95% N-deacetylated and N-sulfate K5 as described hereafter, but in which acetyl groups are normally undetectable with a normal NMR apparatus;        “epiK5”, within the nomenclature of the glucosaminoglycans described herein, designates the K5 and its derivatives in which 20-60% of the glucuronic units are C5-epimerized to iduronic units;        “epiK5-N-sulfate” designates the K5-N-sulfate in which 20-60% of the glucuronic units is C5-epimerized to iduronic units of the type described in WO 92/17507 or WO 01/72848;        “epiK5-amine-O-oversulfate” designates all O-sulfated epiK5-amine with a sulfation degree of at least 3.4;        “N-acyl-epiK5-amine-O-oversulfate” designates an N-acylated epiK5-amine O-oversulfate, with a sulfation degree of at least 3.4;        “K5-amine-O-oversulfate” designates an O-sulfated K5-amine with a sulfation degree of at least 2.2; and        “N-acyl-K5-amine-O-oversulfate” designates an N-acylated N-acyl-K5-amine-O-oversulfate with a sulfation degree of at least 2.2;        
In addition:                the conventional terms and expressions herein defined above refer to K5 as isolated after fermentation, generally with a molecular weight distribution from approximately 1,500 to approximately 50,000 with a mean molecular weight of 10,000-25,000, advantageously of 15,000-25,000;        excepting specific designation of the molecular weight, the conventional terms and expressions herein defined above, when preceded by the acronym “LMW” (low molecular weight), for example LMW-K5-N-sulfate, LMW-epiK5-N-sulfate indicate low molecular weight products, having a mean molecular weight of up to 12,000;        the prefix “(epi)”, which precedes “K5” in conventional terms and expressions as defined herein above, indicates that said K5-N-sulfate, K5-amine-O-oversulfate or N-acyl-K5-O-oversulfate may be non-C5-epimerized or C5-epimerized, namely that said K5-N-sulfate, K5-amine-O-oversulfate or N-acyl-K5-O-oversulfate is formed by mixtures of chains consisting of repetitive sequences of an uronic acid-glucosamine disaccharide of the type (A) above, wherein the uronic units are either all glucuronic acid units (C5-non-epimerized) or K5 polysaccharide) or contain 20-60%, of iduronic acid units (C5-epimerized). Herein below, said prefix (epi) is denoted in the formulae by the symbol “ ” in the 5-position of said uronic unit.        the suffix “-derivative”, which follows the conventional terms and expressions as defined herein above, (a) when added to “(epi)K5-N-sulfate” globally designates (epi)K5-N-sulfates deriving from native K5 polysaccharide and fragments of said (epi)K5-N-sulfates obtained by its depolymerization; (b) when added to “(epi)K5-amine-O-oversulfate” or to “N-acyl-(epi)K5-amine-O-oversulfate”, globally designates (epi)K5-amine-O-oversulfates and N-acyl-(epi)K5-amine-O-oversulfates deriving from (epi)K5-N-sulfate-derivatives; and (c) its absence means that said (epi)K5-N-sulfates, (epi)K5-amine-O-oversulfates or N-acyl-(epi)K5-amine-O-oversulfates either are derived from native K5 polysaccharide or are low molecular weight products but, in this case, the chemical name is preceded by “LMW” as defined above;        the term “approximately”, referring to the molecular weight, designates the molecular weight [± the theoretical weight of a disaccharide unit, including the weight of the sodium, calculated as 461 in the case of an (epi)K5-N-sulfate-derivative] measured by viscosimetry according to Johnson et al., Carbohydrate Res., 1976, 51, 119-127 utilizing samples whose molecular weight was calculated by HPLC as the standard;        the expression “preponderant species”, designates the compound which, in the mixture constituting a lmw-(epi)K5-N-sulfate, a LMW-(epi)K5-amine-oversulfate or a LMW-N-acyl-(epi)K5-amine-O-oversulfate, is the most represented type, determined by the peak of the curve of the molecular weight measured by HPLC;        unless otherwise specifically stated, “degree of sulfation” designates the SO3−/COO− ratio, expressible also as the number of sulfate groups per disaccharide unit, measured with the conductimetric method described by Casu et al., Carbohydrate Res., 1975, 39, 168-176 (“Casu 1975”), the same utilized in WO 01/72848;        “conditions of O-oversulfation” define an extreme O-sulfation performed for example according to the method C described by Casu 1994;        “alkyl” designates a linear or branched alkyd whereas the term “tetrabutylammonium” indicates the tetra(n-butyl)ammonium; and        “functional derivative”, referred to the (C2-C4)Carboxylic acids, defines functional derivatives such as halides; anhydrides; mixed anhydrides; activating esters, for example 2,2,2-trichloroethyl, t-butyl or pentachlorophenyl esters; or the free acid itself, when activated in situ for example with dicyclohexylcarbodiimide.        